PKC-ε regulates vesicle delivery and focal exocytosis for efficient IgG-mediated phagocytosis.

Protein kinase C (PKC)-ε is required for membrane addition during IgG-mediated phagocytosis, but its role in this process is ill defined. Here, we performed high-resolution imaging, which reveals that PKC-ε exits the Golgi and enters phagosomes on vesicles that then fuse. TNF and PKC-ε colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-ε and TNF delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF+ vesicles were not delivered in macrophages from PKC-ε null mice, or upon dissociation of the Golgi-associated pool of PKC-ε, implies that Golgi-tethered PKC-ε is a driver of Golgi-to-phagosome trafficking. Finally, we established that the regulatory domain of PKC-ε is sufficient for delivery of TNF+ vesicles to the phagosome. These studies reveal a novel role for PKC-ε in focal exocytosis - its regulatory domain drives Golgi-derived vesicles to the phagosome, whereas catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain. This article has an associated First Person interview with the first author of the paper.

Bone Marrow-Derived and Elicited Peritoneal Macrophages Are Not Created Equal: The Questions Asked Dictate the Cell Type Used.

Macrophages are a heterogeneous and plastic population of cells whose phenotype changes in response to their environment. Macrophage biologists utilize peritoneal (pMAC) and bone marrow-derived macrophages (BMDM) for in vitro studies. Given that pMACs mature in vivo while BMDM are ex vivo differentiated from stem cells, it is likely that their responses differ under experimental conditions. Surprisingly little is known about how BMDM and pMACs responses compare under the same experimental conditionals. While morphologically similar with respect to forward and side scatter by flow cytometry, reports in the literature suggest that pMACs are more mature than their BMDM counterparts. Given the dearth of information comparing BMDM and pMACs, this work was undertaken to test the hypothesis that elicited pMACs are more responsive to defined conditions, including phagocytosis, respiratory burst, polarization, and cytokine and chemokine release. In all cases, our hypothesis was disproved. At steady state, BMDM are more phagocytic (both rate and extent) than elicited pMACs. In response to polarization, they upregulate chemokine and cytokine gene expression and release more cytokines. The results demonstrate that BMDM are generally more responsive and poised to respond to their environment, while pMAC responses are, in comparison, less pronounced. BMDM responses are a function of intrinsic differences, while pMAC responses reflect their differentiation in the context of the whole animal. This distinction may be important in knockout animals, where the pMAC phenotype may be influenced by the absence of the gene of interest.

Fcγ receptors and toll-like receptor 9 synergize to drive immune complex-induced dendritic cell maturation.

Previous in vivo studies established that inactivated Francisella tularensis immune complexes (mAb-iFt) are a more protective vaccine against lethal tularemia than iFt alone. Subsequent in vitro studies revealed enhanced DC maturation marker expression with mAb-iFt stimulation. The goal of this study was to determine the mechanism of enhanced DC maturation. Multiparameter analysis of surface marker expression and cytokine secretion demonstrates a requirement for FcγR signaling in enhanced DC maturation. MyD88 was also found to be essential for heightened DC maturation, implicating MyD88-dependent TLRs in DC maturation. Upon further study, we discovered that TLRs 2 & 4 drive cytokine secretion, but surprisingly TLR9 is required for DC maturation marker upregulation. These studies reveal a separation of DC cytokine and maturation marker induction pathways and demonstrate that FcγR-TLR/MyD88 synergy underlies the enhanced dendritic cell maturation in response to the mAb-iFt vaccine.